Compositions

ABSTRACT

There is provided inter alia an immunogenic composition comprising a viral antigen,a sugar and/or polyol,an adipate buffer, calcium ions and/or magnesium ions, and one or more positively charged amino acids.

FIELD OF THE INVENTION

The present invention relates to novel viral compositions that areuseful as pharmaceutical compositions and vaccines, to methods forpreparing them and to their use in preventing and treating viralinfections.

BACKGROUND OF THE INVENTION

Acute, infectious diarrhoea is a leading cause of disease and death inmany areas of the world. In developing countries, the impact ofdiarrhoeal disease is very important. For Asia, Africa and LatinAmerica, it has been estimated that there are between 3-4 billion casesof diarrhoea each year and of those cases about 5-10 million result indeath (Walsh, J. A. et al.: N. Engl. J. Med., 301:967-974 (1979)).

Rotaviruses have been recognised as one of the most important causes ofsevere diarrhoea in infants and young children (Estes, M. K. Rotavirusesand Their Replication in Fields Virology, Third Edition, edited byFields et al., Raven Publishers, Philadelphia, 1996). It is estimatedthat rotavirus disease is responsible for over 600,000 deaths annually.Rotavirus-induced illness most commonly affects children between 6 and24 months of age, and the peak prevalence of the disease generallyoccurs during the cooler months in temperate climates, and year-round intropical areas. Rotaviruses are typically transmitted from person toperson by the faecal-oral route with an incubation period of from about1 to about 3 days. Unlike infection in the 6-month to 24-month agegroup, neonates are generally asymptomatic or have only mild disease. Incontrast to the severe disease normally encountered in young children,most adults are protected as a result of previous rotavirus infection somost adult infections are mild or asymptomatic (Offit, P.A. et al. Comp.Ther., 8(8):21-26, 1982).

The development of viral, including rotavirus, formulations must complywith a number of requirements, including worldwide distributionpotential and stability under a broad range of environmental and storageconditions. In particular, the stability of a formulation, especially ofa pharmaceutical or vaccine composition, will in general be better atlower temperatures compared to room or higher temperatures.

Therefore, there is a need for thermostable viral vaccine formulations,including rotavirus,which substantially retain their potency at roomtemperature or higher and which can be afforded by low income anddeveloping world countries. Compositions of the present invention mayhave increased thermostability for a longer duration and/or increasedthermostability at higher temperatures compared to compositions of theprior art.

SUMMARY OF THE INVENTION

The inventors have found that the addition of one or more positivelycharged amino acids to a viral vaccine composition comprising a sugarand/or polyol, an adipate buffer, calcium ions and/or magnesium ions,results in a surprising improvement in thermostability.

Thus, in one aspect, the present invention provides an immunogeniccomposition comprising:

-   -   a viral antigen such as a live attenuated rotavirus,    -   a sugar and/or polyol,    -   an adipate buffer,    -   calcium ions and/or magnesium ions, and    -   one or more positively charged amino acids.

In another aspect, there is provided an immunogenic compositionaccording to the above compositions, for use as a medicament, such asfor use in the treatment or prevention of viral, such as rotavirus,infection; a method for the treatment or prevention of viral, such asrotavirus, infection comprising administering the above immunogeniccomposition to a subject having or at risk of infection; a method forthe preparation of a viral, such as a rotavirus, vaccine comprisingadmixing the above components, and a method of preventing rotavirusinfection and/or rotavirus-caused disease by administering such avaccine to a subject in need thereof.

DESCRIPTION OF THE FIGURES

FIG. 1—Thermostability impact of different buffers, divalent cations andsugars/polyols after 7 days at 45° C.

FIG. 2—Thermostability impact of different buffers, divalent cations andsugars/polyols (subset of data from FIG. 1)

FIG. 3—Thermostability impact of different amino acids on calciumion-containing formulations after 10 days at 45° C.

FIG. 4—Thermostability impact of different amino acids on magnesiumion-containing formulations after 10 days at 45° C.

FIG. 5—Thermostability impact of arginine and/or histidine after storagefor 4, 8 and 10 weeks at 37° C.

FIG. 6—Thermostability impact of various antioxidants and proteins after10 days at 45° C.

FIG. 7—Thermostability of multiple formulations with varied parametersafter 14 days at 45° C.

FIG. 8—Albumin and sucrose relationship after 14 days at 45° C.

FIG. 9—Histidine and sucrose relationship after 14 days at 45° C.

FIG. 10—TPGS and albumin relationship after 14 days at 45° C.

FIG. 11—Thermostability impact of increasing arginine concentrationafter 14 days at 45° C.

FIG. 12—Thermostability impact of varied pH after 14 days at 45° C.

FIG. 13—Thermostability of a formulation comprising 10% w/v sucrose, 5mM calcium ions, 0.2% w/v histidine, 0.2% w/v arginine, 0.2% w/vrecombinant human serum albumin, 1 mM TPGS and having a pH of 6.5, afterstorage at 45° C.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the invention, there is provided an immunogeniccomposition comprising:

-   -   a viral antigen,    -   a sugar and/or polyol,    -   an adipate buffer,    -   calcium ions and/or magnesium ions, and    -   one or more positively charged amino acids.

In a preferred embodiment, the viral antigen is a rotavirus antigen, forexample a live, live attenuated, reassortant or inactivated rotavirus.

Suitably, the viral antigen is a live, live attenuated, reassortant orinactivated virus. In a preferred embodiment, the viral antigen is alive attenuated virus. In a more preferred embodiment, the viral antigenis a live attenuated rotavirus. In a most preferred embodiment, theviral antigen is a human live attenuated rotavirus.

Positively charged amino acids include histidine, arginine and lysine.Preferably, the one or more positively charged amino acids are selectedfrom histidine and/or arginine.

In a preferred embodiment, the immunogenic composition comprises a liveattenuated rotavirus, sucrose, adipic acid, calcium ions and histidine.

In another preferred embodiment, the immunogenic composition comprises alive attenuated rotavirus, sucrose, adipic acid, calcium ions andarginine.

In another preferred embodiment, the immunogenic composition comprises alive attenuated rotavirus, sucrose, adipic acid, magnesium ions andarginine.

In another preferred embodiment, the immunogenic composition comprises alive attenuated rotavirus, sucrose, adipic acid, magnesium ions andhistidine.

Live Attenuated Viral Vaccines

Vaccines comprising live attenuated virus as the antigenic component areknown in the art. Attenuated strains of viruses suitable for use invaccines are known, including strains of adenovirus (e.g., adenovirustype 4, adenovirus type 7), herpes zoster (shingles), measles, mumps,rubella, influenza (e.g., seasonal flu nasal spray, 2009 H1N1 flu nasalspray), varicella (chicken pox), polio, rabies, rotavirus, vaccinia(smallpox) and yellow fever virus. As used herein, a live attenuatedvirus is one that replicates in a suitable host, but where thedisease-causing capacity has been decreased by biological or technicalmethods. Methods of attenuating virus are known in the art, such aspassaging in cell culture, preparing reassortant virus, or using avariant from one species to vaccinate a subject of a different species.Live attenuated virus suitable for use in vaccines are capable ofeliciting a protective immune response in the intended subject.

Rotavirus

Rotaviruses are spherical, and their name is derived from theirdistinctive outer and inner or double-shelled capsid structure.Typically, the double-shelled capsid structure of a rotavirus surroundsan inner protein shell or core that contains the genome. The genome of arotavirus is composed of 11 segments of double-stranded RNA which encodeat least 11 distinct viral proteins. Two of these viral proteinsdesignated as VP4 (P protein) and VP7 (G protein) are structuralproteins arranged on the exterior of the double-shelled capsidstructure. The inner capsid of the rotavirus presents one protein, whichis the rotavirus protein designated VP6. The relative importance ofthese three particular rotavirus proteins in eliciting the immuneresponse that follows rotavirus infection is not yet clear.Nevertheless, the VP6 protein determines the group and subgroup antigen,and VP4 and VP7 proteins are the determinants of serotype specificity.

To date, at least 14 rotavirus G serotypes and 11 rotavirus P serotypeshave been identified (Linhares A. C. & Bresse J. S., Pan. Am. J. Publ.Health 2000, 9, 305-330). Among these, 10 G serotypes and 6 P serotypeshave been identifed among the human rotavirus.

VP7 protein is a 38,000 MW glycoprotein (34,000 MW whennon-glycosylated) which is the translational product of genomic segment7, 8 or 9, depending on the strain. This protein stimulates formation ofthe major neutralising antibody following rotavirus infection. VP4protein is a non-glycosylated protein of approximately 88,000 MW whichis the translational product of genomic segment 4. This protein alsostimulates neutralising antibody following rotavirus infection. SinceVP4 and VP7 proteins are the viral proteins against which neutralisingantibodies are directed, they are believed to be prime candidates fordevelopment of rotavirus vaccines, affording protection againstrotavirus illness.

Natural rotavirus infection during early childhood is known to elicitprotective immunity.

Early vaccine development for preventing rotavirus infections began inthe 1970s after the discovery of the virus. Initially, attenuatedstrains from animals and humans were studied, whilst more recent effortshave focused on human-animal reassortants.

ROTARIX™ is an oral vaccine used for the prevention of rotavirusgastroenteritis caused by G1 and certain non-G1 types; it is a live,attenuated rotavirus vaccine derived from the human 89-12 strain whichbelongs to the G1P[8] type. ROTARIX™ is available as a container oflyophilized vaccine that is reconstituted with a liquid diluent prior tooral application, and is suitable for use in human infants. It isrecommended that both the lyophilized vaccine component and the diluentsbe stored refrigerated at 2° to 8° C. (36° to 46° F.).

It is known that many vaccines must be stored and transported atrefrigeration temperatures maintained at 2° C. to 8° C. Further, it isalso known that many vaccines must be administered immediately on beingremoved from refrigeration. This necessitates strict cold chain storageand transport which is problematic particularly in the developing andlow income regions where the cold chain required for maintaining vaccinepotency and efficacy is imperfect, overburdened or nonexistent,resulting in vaccine being wasted.

One thermostabilisation method has been to develop vaccine formulationsthat can be stored frozen (−20° C. to −70° C.) or alternatively todevelop lyophilised vaccines that can be kept for a prolonged period oftime at around refrigerator temperature (2° C. to 8° C.). However, it isa known fact that the lyophilisation process has a limiting capacity,and is associated with a high production cost. Furthermore,administering lyophilised vaccines may require more complex, hencerelatively expensive, devices such as multichamber/vial vaccines, withthe active ingredient in one chamber and the reconstitution liquid inanother chamber. Lyophilised vaccines are also associated with highershipment and storage cost. These options may be inadequate for somecountries in the developing world where the administration device has tobe affordable and where the availability of production and storageinfrastructure may be non existent or unreliable.

Improving rotavirus vaccine formulation thermostability could impact allthree of the following goals (Karp et al., Vaccine 201533(30):3471-3479): (i) development of fully thermostable vaccines couldincrease coverage by enabling the stocking of vaccines at facilitiesthat do not have cold chain equipment and by facilitating outreach; (ii)the development of such vaccines might improve efficacy by decreasingthe probability of administering vaccines whose efficacy was impaired byheat and/or freeze exposure and (iii) total system costs could bereduced by decreasing vaccine wastage due to detected heat and freezeexposures, by decreasing the cold chain footprint, and by reducing theoverall requirements for the vaccine delivery supply chain.

International patent application number WO2009042202 disclosesformulations for stabilisation of rotavirus and methods for stabilisingviruses in liquid and dried formulations. In particular, formulationsare provided including Zn²⁺ cations which may stabilise the viability ofrotaviruses.

Composition Components

Rotavirus

The composition of the present invention comprises a viral antigen,preferably a virus, such as a rotavirus. In particular the compositionis an immunogenic composition, e.g. a vaccine composition. A rotavirusis understood to mean any rotavirus that is suitable for use in avaccine formulation, e.g., a vaccine for administration to humans. Orallive rotavirus are especially contemplated. For example, any suitablerotavirus can be selected from the group consisting of: a liveattenuated rotavirus from animals or humans, in particular a human liveattenuated rotavirus; a reassortant rotavirus, in particular but notlimited to a human-human reassortant rotavirus, a bovine-humanreassortant rotavirus or a rhesus monkey-human reassortant rotavirus.

All rotavirus strains, human or animal strains, are contemplated in thepresent invention. Human rotavirus strains are especially suitable. Inparticular, the rotavirus is in one embodiment an attenuated humanrotavirus, and the composition comprises a single variant orsubstantially a single variant, said variant being defined by thenucleotide sequence encoding at least one of the major viral proteinsdesignated as VP4 and VP7 as disclosed in WO 01/12797, in particularany, including one or more, of the variants defined by the mutations setforth in Table 2, Tables 3.1 and 3.2 of WO 01/12797. In specificembodiments, the rotavirus antigen is any of the following liveattenuated human rotavirus (HRV) strains: HRV 89-12C2 strain depositedunder accession number ATCC VR 2272 (as described in EP 0 557 427), itsprogeny, reassortants and immunologically active derivatives thereof;HRV P43 strain deposited under accession number ECACC 99081301 (asdescribed in WO 01/12797), its progeny, reassortants and immunologicallyactive derivatives thereof. Suitably the rotavirus is present in thecomposition of the invention at a titre ranging from about 1×10⁵ toabout 1×10⁸ pfu/mL.

Rotavirus belonging to strains which have the characteristics of any ofthe above mentioned deposited strains are also suitable vaccine strains.Derivatives from said deposited strains can be obtained by subjectingsaid strains to further processing such as by propagating them byfurther passaging, cloning, or other procedures using the live virus orby modifying said deposited strains in any way including by geneticengineering techniques or reassortant techniques. Such steps andtechniques are well known in the art. Rotavirus of particular interestare progeny of any of said deposited strains and immunologically activederivatives thereof. Immunologically active derivatives means materialsobtained from or with any of the deposited strains, in particular fromor with HRV P43 strain deposited under accession number

ECACC 99081301.

Of particular interest are reassortant rotaviruses which comprise atleast one antigen or at least one segment of any of said depositedstrains, for example reassortants which comprise a virulent strain ofrotavirus in which one or part of one of the 11 genome segments has beenreplaced by the genome segment or part thereof of any of said depositedstrains. Specifically, a rotavirus reassortant in which the segment orpartial segment coding for NSP4 is a segment or partial segment of anyof said deposited strains, may have useful properties. Reassortantrotaviruses and techniques for preparing them are well known (Foster, R.H. and Wagstaff, A. J. Tetravalent Rotavirus Vaccine, a review. ADISdrug evaluation, BioDrugs, Gev, 9 (2), 155-178, 1998).

The rotavirus used in the compositions of the present invention may beproduced according to routine production techniques. Typically rotavirusantigen preparations may be derived from tissue culture methods used topropagate the virus. Suitable cell substrates for growing the virusinclude for example dog kidney cells such as MDCK or cells from a cloneof MDCK, MDCK-like cells, monkey kidney cells such as AGMK cellsincluding Vero cells which are particularly suitable, other cells linesof monkey kidney origin such as BSC-1, LLC-MK2 and MA104, suitable pigcell lines, or any other mammalian cell type suitable for the productionof rotavirus for vaccine purposes. Suitable cell substrates also includehuman cells e.g. MRC-5 cells. Suitable cell substrates are not limitedto cell lines; for example primary cells are also included.

Also within the scope of the invention are admixtures of any of theabove recited deposited strains with other rotavirus variants, forexample other cloned variants or other reassortant rotavirus, or withother viruses in particular other attenuated viruses.

The rotavirus for inclusion in the compositions of the present inventioncan be a monovalent rotavirus strain, i.e. containing a single rotavirusstrain, or be multivalent, i.e. containing at least two or morerotavirus strains.

Suitably the rotavirus is derived from the G1P[8] type. More suitably,the rotavirus is derived from the human 89-12 strain.

A particularly suitable immunogenic composition contains the attenuatedHRV P43 strain (“RIX4414”, as deposited under accession number ECACC99081301, see WO 01/12797), suitably at a concentration of 10⁵-10⁶ ffu(focus forming units) per dose (or equivalent to 10^(5.5)-10^(6.5) asexpressed in CCID₅₀ (median Cell Culture Infective Dose) per dose).

The skilled person will understand that other readily availableattenuated strains, from human or animal origin, that are obtainablefrom depository institutions are also suitable and may be used assubstitutes for the recited deposited strains.

Positively Charged Amino Acid

Compositions according to the invention comprise one or more positivelycharged amino acids.

Positively charged amino acids include histidine, arginine and lysine.

Histidine (abbreviated as His or H; encoded by the codons CAU and CAC)is an α-amino acid that is used in the biosynthesis of proteins. Itcontains an α-amino group (which is in the protonated —NH³⁺ form underbiological conditions), a carboxylic acid group (which is in thedeprotonated —COO⁻ form under biological conditions), and a side chainimidazole, classifying it as a positively charged (at physiological pH)amino acid. Suitably, histidine salts, enantiomers and other variants ofhistidine may be used.

Arginine (abbreviated as Arg or R) encoded by the codons CGU, CGC, CGA,CGG, AGA, and AGG is an α-amino acid that is used in the biosynthesis ofproteins. It contains an α-amino group (which is in the protonated —NH³⁺form under biological conditions), an α-carboxylic acid group (which isin the deprotonated —COO⁻ form under biological conditions), and a sidechain of a 3-carbon aliphatic straight chain capped by a complexguanidinium, classifying it as a charged (at physiological pH)aminoacid. Suitably, arginine salts, enantiomers and other variants ofarginine may be used.

Lysine (abbreviated as Lys or K; encoded by the codons AAA and AAG) isan α-amino acid that is used in the biosynthesis of proteins. Itcontains an α-amino group (which is in the protonated —NH³⁺ form underbiological conditions), an α-carboxylic acid group (which is in thedeprotonated —COO⁻ form under biological conditions), and a side chainlysyl ((CH₂)4NH₂), classifying it as a positively charged (atphysiological pH), aliphatic amino acid. Suitably, lysine salts,enantiomers and other variants of lysine may be used.

Suitably the one or more positively charge amino acid are present at aconcentration of at least 0.01% w/v, more suitably at least 0.05% w/v,more suitably at least 0.1% w/v, more suitably at least 0.15% w/v, moresuitably at least 0.2% weight/volume (w/v). Suitably the one or morepositively charge amino acid are present at a concentration of about orexactly 0.2% w/v.

Suitably the one or more positively charge amino acid are present at aconcentration from about 0.01% w/v, 0.05% w/v, or 0.1% w/v, to about 1%w/v, 0.5% w/v, or 0.3% w/v. Suitably the histidine is present at aconcentration of from about 0.01% to about 1% w/v, more suitably fromabout 0.05% to about 0.5% w/v, more suitably from about 0.1% w/v toabout 0.3% w/v.

In a preferred embodiment, the positively charged amino acid areselected from histidine and/or arginine.

Suitably the histidine is present at a concentration of at least 0.01%w/v, more suitably at least 0.05% w/v, more suitably at least 0.1% w/v,more suitably at least 0.15% w/v, more suitably at least 0.2%weight/volume (w/v). Suitably the histidine is present at aconcentration of about or exactly 0.2% w/v.

Suitably the histidine is present at a concentration from about 0.01%w/v, 0.05% w/v, or 0.1% w/v, to about 1% w/v, 0.5% w/v, or 0.3% w/v.Suitably the histidine is present at a concentration of from about 0.01%to about 1% w/v, more suitably from about 0.05% to about 0.5% w/v, moresuitably from about 0.1% w/v to about 0.3% w/v.

Suitably the arginine is present at a concentration of at least 0.01%w/v, more suitably at least 0.05% w/v, more suitably at least 0.1% w/v,more suitably at least 0.15% w/v, more suitably at least 0.2% w/v.Suitably the arginine is present at a concentration of about or exactly0.2% w/v.

Suitably the arginine is present at a concentration from about 0.01%w/v, 0.05% w/v, or 0.1% w/v, to about 1% w/v, 0.5%w/v, or 0.3% w/v.Suitably the arginine is present at a concentration of from about 0.01%w/v to about 1% w/v, more suitably from about 0.05% w/v to about 0.5%w/v, more suitably from about 0.1% w/v to about 0.3% w/v.

Suitably the composition comprises both histidine and arginine, suitablywherein each is present in any one of the concentration ranges specifiedabove (i.e., the concentration of arginine and histidine may be the sameor may differ within a composition).

Calcium Ions and/or Magnesium Ions

The composition according to the invention comprises calcium ions and/ormagnesium ions.

Suitably the calcium ions are present at a concentration from about 0.5mM, 1 mM, 3 mM or 5 mM, to about 7 mM, 15 mM, or 7 mM. Suitably thecalcium ions are present at a concentration of from about 0.5 mM toabout 20 mM, more suitably from about 1 mM to about 15 mM, more suitablyfrom about 3 mM to about 7 mM, more suitably about or exactly 5 mM.

Suitably the calcium ions are present at a concentration of at least 1mM, more suitably at least 3 mM, more suitably at least 4 mM, moresuitably at least 5 mM.

Suitably the calcium ions are present in the form of calcium chloride.

Suitably when the composition of the invention comprises calcium ions,the composition also comprises histidine.

Suitably the magnesium ions are present at a concentration from about0.5 mM, 1 mM, 3 mM or 5 mM, to about 7 mM, 15 mM, or 7 mM. Suitably themagnesium ions are present at a concentration of from about 0.5 mM toabout 20 mM, more suitably from about 1 mM to about 15 mM, more suitablyfrom about 3 mM to about 7 mM, more suitably about or exactly 5 mM.

Suitably the magnesium ions are present at a concentration of at least 1mM, more suitably at least 3 mM, more suitably at least 4 mM, moresuitably at least 5 mM.

Suitably the magnesium ions are present in the form of magnesiumsulphate.

Suitably when the composition of the invention comprises magnesium ions,the composition also comprises arginine.

Suitably the composition comprises both calcium ions and magnesium ions,each in any one of the concentration ranges specified above (i.e., theconcentration of arginine and histidine may be the same or may differwithin a composition).

Suitably the composition comprises zinc ions. Suitably the zinc ions arepresent at a concentration of about 0.5 mM to about 20 mM. Suitably thezinc ions are present at a concentration of lower than 0.1 mM. Suitablythe composition is substantially free of zinc ions.

Adipate Buffer

The composition according to the invention comprises an adipate buffer.Suitably the adipate buffer is a salt of adipic acid, e.g., monosodiumsalt of adipic acid, monopotassium salt of adipic acid, suitablydisodium adipate or dipotassium adipate, or calcium adipate. Moresuitably, the adipate buffer is di-sodium adipate.

Suitably, the composition comprises adipate buffer at about 1% w/v, 3%w/v, 5% w/v or 7% w/v w/v, to about 8% w/v, 10% w/v, or 20% w/v, forexample, from about 3% to about 10% w/v, more suitably about 5% to about8% w/v, more suitably about 7% w/v adipate buffer.

It will be understood that the adipate concentration within the rangementioned above may be suitably adapted, through routineexperimentation, according to the antacid capacity to be achieved andthe volume of the vaccine dose.

Sugar/Polyol

The composition of the invention comprises one or more sugar(s) and/orpolyol(s). Suitably the sugar(s) and/or polyol(s) is selected from thelist consisting of: glycerol, erythrose, fucose, erythriol, xylitol,arabitol, ribose, dextrose, xylose, arabinose, glucose, tagalose,mannose, galactose, fructose, inositol, sorbitol, mannitol, galactitol,a combination of glucose and fructose, maltose, sophorose, lactose,cellobiose, melibiose, trehalose, sucrose, palatinose, maltulose,lactulose, maltitol, lactitol, raffinose, maltotriose, melezitose,cellotriose, ciritol, maltotetraose, stachyose, cellotetraose,maltopentaose, cellopentaose, maltohexaose, cellohexaose, dextransulphate and α-cyclodextrin.

More suitably the sugar(s) and/or polyol(s) is selected from the listconsisting of: sucrose, glucose, maltose, trehalose, fructose,α-cyclodextrin, sorbitol and dextran sulphate. More suitably the sugarand/or polyol is sucrose or sorbitol. More suitably the sugar and/orpolyol is a sugar. More suitably the sugar is sucrose.

Suitable sugar and/or polyol concentrations in the composition of theinvention may range from about 1% w/v, 3% w/v, 4% w/v, 5% w/v, 6% w/v,7% w/v, 8% w/v, to about 10% w/v, 13% w/v, 20% w/v, 30% w/v, 35% w/v,40% w/v, or about 70% w/v, for example from about 3% w/v to about 40%w/v, more suitably from about 4% w/v to about 35% w/v, more suitablyfrom about 5% w/v to about 30% w/v, more suitably from about 6% w/v toabout 20% w/v, more suitably from about 7% w/v to about 15% w/v, moresuitably from about 8% w/v to about 13% w/v, more suitably about 10%w/v.

Suitably the concentration of the sugar and/or polyol is no more than70% w/v, more suitably no more than 50% w/v, more suitably no more than40% w/v, more suitably no more than 35% w/v, more suitably no more than30% w/v, more suitably no more than 25% w/v, more suitably no more than20% w/v, more suitably no more than 15% w/v, more suitably no more than10% w/v.

pH

Suitably the pH of the composition is from about 5.0, 5.5, 5.7, 6.0,6.3, 6.4 or 6.5, to about pH 6.6, 6.7, 7.0, 7.3, 7.5, or 8.0 for examplebetween pH 5.0 and pH 8.0, more suitably between about pH 5.5 to aboutpH 7.5, more suitably between about pH 5.7 to about pH 7.3, moresuitably between about pH 6.0 to about pH 7.0, more suitably betweenabout pH 6.3 to about pH 6.7, more suitably between about pH 6.4 toabout pH 6.6, more suitably about 6.5.

The pH of the rotavirus immunogenic composition as described herein maybe obtained by mixing of adipic acid and a carboxylate salt.Alternatively, the pH of the rotavirus immunogenic composition asdescribed herein may be obtained by mixing of adipic acid and sodiumhydroxide. In particular, the adipic acid may be used in admixture witha different carboxylate salt, for example, a citrate is combined withadipic acid. This may be advantageous when using commercially availablechemicals, some of which may not be readily available, or to simplifythe formulation step. The skilled person will appreciate thatformulation excipients may have an impact on the pH of a formulation.

Proteins/Antioxidants

Suitably the composition comprises one or more proteins or antioxidantsselected from the list consisting of vitamin E succinate (VES),D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS), heparin,monothioglycerol (MTG), lactalbumin, albumin and β casein. More suitablythe composition comprises TPGS and/or albumin, more suitably both TPGSand albumin.

Suitably the albumin is human serum albumin, more suitably recombinanthuman serum albumin.

Suitably albumin is present in the composition at a concentration of atleast 0.05% w/v, more suitably at least 0.1% w/v, more suitably at least0.15% w/v, more suitably at least 0.2% w/v. Suitably albumin is presentin the composition at a concentration of no more than 0.2% w/v.

Suitably vitamin E succinate or TPGS is present in the composition at aconcentration of at least 0.5 mM, more suitably at least 1 mM, moresuitably at least 1.5 mM, more suitably at least 2 mM. Suitably vitaminE succinate or TPGS is present in the composition at a concentration ofno more than 2 mM, more suitably no more than 1.5 mM, more suitably nomore than 1 mM. Suitably vitamin E succinate or TPGS is present in thecomposition at a concentration of about or exactly 1 mM.

Other Components

The composition according to the present invention may further include aphosphate. Suitably the phosphate is selected from the group consistingof: monophosphates, polyphosphates and phosphorylated compounds.Phosphate refers as the salt of phosphoric acid (also known asorthophosphoric acid (H₃PO₄)), usually sodium or potassium or a mix ofsodium and potassium salts are used (for example: Na₃PO₄, Na₂HPO₄,NaH₂PO₄, K₃PO₄, K₂HPO₄, KH₂PO₄). Suitably, the phosphate concentrationis from about 0.05 to about 0.3 M. Typically phosphate, when present,comes from the cell culture medium or saline buffer used as a diluent,such as DMEM (Dulbecco's modified Eagle Medium), Eagle BME basal mediumor phosphate-buffered saline (PBS). Suitably the phosphate is present ata concentration of from about 10 mM to about 2 M.

Suitably the composition of the invention is an aqueous solution, moresuitably an aqueous saline solution.

Suitably the composition of the invention comprises a non-ionicsurfactant. Suitably the non-ionic surfactant is selected from the groupconsisting of polysorbates, polyoxyethylene alkyl ether, nonaethyleneglycol octylphenyl ether, hepatethylene glycol octylphenyl ether,sorbitan trioleate, and polyoxyethylene-polyoxypropylene blockcopolymer. Suitably the non-ionic surfactant concentration is from about0.005% w/v to about 0.1% w/v.

The composition according to the present invention may further includean additional antacid component such as an inorganic antacid, forexample aluminium hydroxide Al(OH)₃ and/or magnesium hydroxide Mg(OH)₂.Aluminium hydroxide is particularly suitable. Other commerciallyavailable antacids, which are suitable for use in the invention, includeMYLANTA™, which contains aluminium hydroxide and magnesium hydroxide.These are insoluble in water and are given in suspension. Anotherparticularly suitable antacid that may be additionally used in thevaccine composition of the present invention is the insoluble inorganicsalt, calcium carbonate (CaCO₃). A typical CaCO₃ concentration is 80 mgper vaccine dose for example.

Other suitable water insoluble antacids are magnesium carbonate,aluminium carbonate, aluminium phosphate, mix of aluminium hydroxide andmagnesium carbonate, aluminium-magnesium-hydrycarbonate, aluminiumhydroxide-magnesium carbonate-sorbitol-manitol,hydroxy-aluminium-sodium-carbonate,dihydroxy-aluminium-potassium-carbonate, magaldrate, hydrotalcite,almagcit and magnesium-aluminium-silicate-hydrate.

The composition according to the present invention may additionallycomprise a pharmaceutically acceptable excipient and/or carrier, inparticular those known in the art as being suitable for oraladministration, especially to humans and more especially to humaninfants. Such carriers include carbohydrates, polyalcohols,hydroxyapatite, talc, titanium oxide, iron hydroxide, magnesiumstearate, carboxymethylcellulose, hydroxypropylmethylcellulose,microcrystalline cellulose, gelatin, vegetal peptone, xanthane,caraghenane, arabic gum and β-cyclodextrin.

Suitably the composition of the invention comprises a diluent. Suitablythe diluent is selected from the group consisting of: a tissue culturemedium, saline, phosphate-buffered saline and water.

Viscous agents may additionally be included in the composition. Viscousagents that may be used include pseudoplastic excipients. Suitableviscous agents include: propylene glycol, arabic gum, adragant gum,agar-agar, alginate, pectin, sodium carboxymethylcellulose (TYLOSES C™),methylcellulose (METHOCELS A™, VISCOTRANS MC™, TYLOSE MH™ and MB™),hydroxypropylmethylcellulose (KLUCELS™), hydroxypropylcellulose(METHOCELS E™ and K™, VICOTRANS MPHC™), CARBOPOL™, xanthane gum, VEEGUM™(Magnesium-aluminium silicate), AVICEL™ (about 89% microcrystallinecellulose and 11% Carboxymethylcellulose Na). Xanthane gum or starch areparticularly suitable viscous agents for use in the compositionaccording to the invention.

It may also be advantageous to include in the compositions of thepresent invention lipid-based vehicles such as virosomes or liposomes,oil-in-water emulsions or carrier particles. Alternatively or inaddition immunostimulants such as those known in the art for oralvaccines may be included in the composition. Such immunostimulantsinclude bacterial toxins, particularly cholera toxin (CT) in the form ofthe holotoxin (entire molecule) or the B chain only (CTB) and the heatlabile enterotoxin of E. coli (LT). Mutated LTs (mLTs) which are lesslikely to convert to their active form than the native LT are describedin WO 96/06627, WO 93/13202 and U.S. Pat. No. 5,182,109.

The composition according to the invention may further comprise anadjuvant or immunostimulant such as but not limited to detoxified lipidA from any source and non-toxic derivatives of lipid A, saponins andother reagents capable of stimulating a TH1 type response.

It has long been known that enterobacterial lipopolysaccharide (LPS) isa potent stimulator of the immune system, although its use in adjuvantshas been curtailed by its toxic effects. A non-toxic derivative of LPS,monophosphoryl lipid A (MPL), produced by removal of the corecarbohydrate group and the phosphate from the reducing-end glucosamine,has been described by Ribi et al (1986, Immunology andImmunopharmacology of bacterial endotoxins, Plenum Publ. Corp., N.Y.,p407-419).

A further detoxified version of MPL results from the removal of the acylchain from the 3-position of the disaccharide backbone, and is called3-O-Deacylated monophosphoryl lipid A (3D-MPL). It can be purified andprepared by the methods taught in GB 2122204B, which reference alsodiscloses the preparation of diphosphoryl lipid A, and 3-O-deacylatedvariants thereof.

A suitable form of 3D-MPL is in the form of an emulsion having a smallparticle size less than 0.2 μm in diameter, and its method ofmanufacture is disclosed in WO 94/21292. Aqueous compositions comprisingmonophosphoryl lipid A and a surfactant have been described inWO9843670A2.

The bacterial lipopolysaccharide derived adjuvants to be formulated inthe compositions of the present invention may be purified and processedfrom bacterial sources, or alternatively they may be synthetic. Forexample, purified monophosphoryl lipid A is described in Ribi et al 1986(supra), and 3-O-Deacylated monophosphoryl or diphosphoryl lipid Aderived from Salmonella sp. is described in GB 2220211 and U.S. Pat. No.4,912,094. Other purified and synthetic lipopolysaccharides have beendescribed (Hilgers et al., 1986, Int.Arch.Allergy.Immunol., 79(4):392-6;Hilgers et al., 1987, Immunology, 60(1):141-6; and EP 0 549 074 B1). Aparticularly suitable bacterial lipopolysaccharide adjuvant is 3D-MPL.

Accordingly, the LPS derivatives that may be used in the presentinvention are those immunostimulants that are similar in structure tothat of LPS or MPL or 3D-MPL. In another aspect of the present inventionthe LPS derivatives may be an acylated monosaccharide, which is asub-portion to the above structure of MPL.

Synthetic derivatives of lipid A are also known including, but notlimited to:

OM 174(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o-phosphono-β-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranosyldihydrogenphosphate),(WO 95/14026)

OM 294 DP (3S, 9R)-3--[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate)(WO99/64301 and WO 00/0462)

OM 197 MP-Ac DP (3S-,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1-dihydrogenophosphate10-(6-aminohexanoate) (WO 01/46127)

Purified saponins as oral adjuvants are described in WO 98/56415.Saponins and monophosphoryl lipid A may be employed separately or incombination (e.g. as described in WO 94/00153) and may be formulated inadjuvant systems together with other agents. 3D-MPL is a well-knownadjuvant manufactured by Corixa Corporation, Montana and its manufactureis described in GB 2122204.

Another prefered immunostimulant for use in the present invention isQuil A saponin and its derivatives. Saponins are taught in:Lacaille-Dubois, M and Wagner H. (1996. A review of the biological andpharmacological activities of saponins. Phytomedicine vol 2 pp 363-386).Saponins are steroid or triterpene glycosides widely distributed in theplant and marine animal kingdoms. Saponins are noted for formingcolloidal solutions in water which foam on shaking, and forprecipitating cholesterol. When saponins are near cell membranes theycreate pore-like structures in the membrane which cause the membrane toburst. Haemolysis of erythrocytes is an example of this phenomenon,which is a property of certain, but not all, saponins.

Saponins are known as adjuvants in vaccines for systemic administration.The adjuvant and haemolytic activity of individual saponins has beenextensively studied in the art (Lacaille-Dubois and Wagner, supra). Forexample, Quil A (derived from the bark of the South American treeQuillaja Saponaria Molina), and fractions thereof, are described in U.S.Pat. No. 5,057,540 and “Saponins as vaccine adjuvants”, Kensil, C. R.,Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279B1. Particulate structures, termed Immune Stimulating Complexes(ISCOMS), comprising fractions of Quil A are haemolytic and have beenused in the manufacture of vaccines (Morein, B., EP 0 109 942 B1; WO96/11711; WO 96/33739). The haemolytic saponins QS21 and QS17 (HPLCpurified fractions of Quil A) have been described as potent systemicadjuvants, and the method of their production is disclosed in U.S. Pat.No.5,057,540 and EP 0 362 279 B1. QS-21 is a natural saponin derivedfrom the bark of Quillaja saponaria Molina, which induces CD8+ cytotoxicT cells (CTLs), Th1 cells and a predominant IgG2a antibody response andis a suitable saponin in the context of the present invention. Othersaponins which have been used in systemic vaccination studies includethose derived from other plant species such as Gypsophila and Saponaria(Bomford et al., Vaccine, 10(9):572-577, 1992).

An enhanced system involves the combination of a non-toxic lipid Aderivative and a saponin derivative particularly the combination of QS21and 3D-MPL as disclosed in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol as disclosed inWO 96/33739. The saponins may be separate in the form of micelles, ormay be in the form of large ordered structures (such as ISCOMs (EP 0 109942 B1) or liposomes) when formulated with cholesterol and lipid, or inthe form of an oil-in-water emulsion (WO 95/17210). The saponins maysuitably be associated with a metallic salt, such as aluminium hydroxideor aluminium phosphate (WO 98/15287).

Potent adjuvant compositions comprising QS21 and 3D-MPL in anoil-in-water emulsion are described in WO 95/17210 and in WO 99/11241and WO 99/12565, and are suitable compositions.

A general discussion of vehicles and adjuvants for oral immunisation canbe found in Vaccine Design, The Subunit and Adjuvant Approach, edited byPowell and Newman, Plenum Press, N.Y., 1995.

The vaccine composition according to the invention may containadditional components including for example flavourings (particularlyfor an oral vaccine) and bacteriostatic agents.

Additional Antigens

The composition of the invention may also be formulated to containfurther, additional antigens, in particular antigens from other suitableviruses for protection against other diseases, for example poliovirus.Said additional antigens may be given either in admixture with therotavirus composition, or alternatively may be co-administered (i.e. ina separate dose but on the same occasion) with the rotaviruscompositions of the present invention.

The compositions of the present invention may also be givenconcomitantly with other (for example, non-oral) vaccines, for examplewith parenteral vaccines suitable for the paediatric vaccinee populationsuch as DTPw or DTPa vaccines (vaccines against Bordetellapertussis—whooping cough, diphteria, tetanus), vaccines againstHaemophilus influenza B-induced meningitis, hepatitis B, or measles,mumps, rubella (MMR), vaccines against Streptococcus pneumoniae, inorder to reduce the number of visits to the doctor.

Particular Formulations

Suitably the composition according to the invention comprises thefollowing:

-   -   a virus, such as a rotavirus, at a titre ranging from about        1×10⁵ to about 1×10⁸ pfu/mL,    -   sucrose at a concentration of 5% w/v to 35% w/v,    -   adipate buffer at a concentration of 3% to about 10% w/v,    -   calcium ions at a concentration of 3 mM to about 7 mM,    -   histidine at a concentration of at least 0.05% w/v.

Alternatively, the composition of the invention may comprise thefollowing:

-   -   a virus, such as a rotavirus, at a titre ranging from about        1×10⁵ to about 1×10⁸ pfu/mL,    -   sucrose at a concentration of 5% w/v to 35% w/v,    -   adipate buffer at a concentration of 3% to about 10% w/v,    -   calcium ions at a concentration of 3 mM to about 7 mM,    -   arginine at a concentration of at least 0.05% w/v.

Thermostability

The thermostability of a viral composition, such as a rotaviruscomposition may be assessed by measuring the virus titer of thecomposition, then storing the composition at a set temperature for a setperiod of time, followed by measuring the virus titer loss relative tothe virus titer before storage, expressed in log₁₀ ffu/mL. Thus, as usedherein, stability of a virus or a viral formulation refers to theability to resist or slow decreases in viral titer over time;thermostability refers to the ability to resist or slow decreases inviral titer due to heat (e.g., storage at non-refrigeratedtemperatures).

Suitably the composition of the invention has a level of thermostabilitysuch that, after storage of the composition for 2.5 months at 37° C.,the composition has a maximum virus titer loss of 1.5, more suitably1.4, more suitably 1.3, more suitably 1.2; as expressed in log₁₀ ffu/mL(loss as compared to the composition's initial rotavirus titer, beforestorage).

Suitably the composition of the invention has a level of thermostabilitysuch that, after storage of the composition for seven weeks at 40° C.,the composition has a maximum rotavirus titer loss of 2.0, more suitably1.5, more suitably 1.4, more suitably 1.3, more suitably 1.2; asexpressed in log₁₀ ffu/mL (loss as compared to the composition's initialrotavirus titer, before storage).

A suitable method for measuring log₁₀ ffu/mL is as follows. Sampledilutions are inoculated on a MA-104 lawn cell for 16-18 hours at 37°C.±1° C. to allow for a viral replication cycle. Viral particles arerevealed by immunoluminescence. After incubation, viral particles aredetected by monoclonal (9F6) antibody (against VP6) which is furtherrevealed by a second antibody (anti-mouse) coupled with HRP. Trubluereagent is then added and turned into blue dots. Rotavirus identity isconfirmed by using highly specific 9F6 monoclonal antibody against VP6.The viral titre is obtained by counting the blue dots and may beexpressed in FFU/ml or FFU/dose.

A suitable method for measuring CCID₅o is as follows. Sample dilutionsare inoculated on a MA-104 lawn cell for 7 days±1 day at 37° C.±1° C.Viral particles are detected by immunofluorescence method. Afterincubation, infected cells come into contact with anti-rotavirus 2C9monoclonal antibody (against VP7) which is further revealed by a secondantibody (anti-mouse) coupled with fluorescence molecule(FITC=Fluorescein IsoThioCyanate). Observation of fluorescent cellsunder the microscope indicates that the cell lawn was well infected bythe rotavirus. Rotavirus identity is confirmed by using highly specific2C9 monoclonal antibody against VP7. The viral titre is obtained by theReed and Muench calculation method (Reed and Muench (1938) The AmericanJournal of Hygiene 27:493-497) and may be expressed in CCID₅₀/ml orCCID₅₀/dose.

Administration

The terms ‘formulation’ and ‘composition’ are used interchangeablyherein. Suitably the formulation of the invention is a vaccine. Theformulation may be provided in the form of a liquid formulation (such asa liquid suspension) or a dry formulation (such as a powder, adissolving tablet for dissolution when placed in the mouth, or an oralthin film).

Suitably the composition according to the invention is administered byoral administration, suitably to humans and more suitably to humaninfants. Suitably the composition is supplied in a single-dose device,such as a glass or plastic vial or syringe, suitable for delivery toinfants.

The composition according to the present invention is an immunogeniccomposition, e.g. it is suitably a vaccine. As used herein, a vaccinerefers to an immunogenic composition capable, after a suitable dosingregime in a subject, of eliciting an immune response in that subjectspecific to an antigen contained in the vaccine. A vaccine will reducethe occurrence or incidence of infection and/or disease (specific to thevaccine's target pathogen) in an appropriately treated population.

As used herein, a rotavirus vaccine is an immunogenic compositioncapable, typically after one, suitably two doses separated by one or twomonths, of eliciting an immune response to rotaviral antigen(s)contained in the vaccine, e.g. a serum rotavirus specific IgA response.As used herein a vaccine also demonstrates an acceptable ‘vaccine take,’defined as the percentage of subjects displaying either a serologicalresponse, e.g. appearance of serum IgA to rotavirus in post-immunizationsera at a titer ≥20 U/ml (ELISA), and/or with rotavirus shedding (ELISA)in any stool sample. Vaccine take can be defined as vaccine virusshedding in any stool sample collected between the first dose and up to1 to 2 months after the second dose.

In one aspect of the invention there is provided the use of theimmunogenic composition of the invention in the manufacture of amedicament for use in the treatment or prevention of viral infection. Inone aspect of the invention the immunogenic composition is a rotavirusvaccine administered to humans, including human infants, and theinfection is a rotavirus infection that affects humans.

In a further aspect of the invention there is provided a method for theprevention of rotavirus infection, and/or rotavirus-associated diseasesuch as gastroenteritis and/or diarrhea, in humans, including humaninfants, where the method comprises administering a rotavirus vaccinecomposition of the invention to a subject at risk of rotavirusinfection.

In a specific embodiment, the rotavirus vaccine according to theinvention is capable of decreasing the risk of, or the occurrence ofany, and preferably severe, rotavirus gastroenteritis as compared toplacebo. Typically the vaccine is able to confer cross-protectionagainst circulating strains of rotavirus other than that present in thevaccine. Typically, when the vaccine contains a G1 type strain such asthat of the attenuated human virus P43, an immune response is induced toG1 and at least one of the non-G1 serotypes selected from the groupconsisting of: G2, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12, G13 andG14 serotypes. Suitably a vaccine containing a G1 strain is capable ofconferring protection against both G1 and non-G1 strains, such as G2, G3and/or G4 strains, and in particular against the globally emerging G9serotype.

In a specific embodiment, the rotavirus infection causes gastroenteritisor severe gastroenteritis. In one embodiment, the gastroenteritis orsevere gastroenteritis is caused by a rotavirus strain of the sameserotype to that contained in the rotavirus vaccine composition of theinvention.

In a further embodiment, the gastroenteritis or severe gastroenteritisis caused by a rotavirus strain of a different serotype to thatcontained in the rotavirus vaccine composition of the invention. Inparticular, if the rotavirus strain present in the composition is a G1serotype, such as but not limited to the live attenuated human rotavirusstrain HRV P43 (ECACC 99081301), prevention is conferred againstgastroenteritis or severe gastroenteritis caused by a rotavirus strainof a G1 serotype and also by a rotavirus strain of a non-G1 serotype,for example by a rotavirus strain having a serotype selected from thelist consisting of: G2, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12, G13and G14. In a particular embodiment, the immunogenic composition iscapable of inducing an immune response against, and/or provideprotection against gastroenteritis or severe gastroenteritis caused by,at least one, suitably all of the following non-G1 serotypes: G2, G3, G4and G9. In another specific embodiment, if the rotavirus strain presentin the composition of the present invention is a P[8] rotavirus type,such as but not limited to the live attenuated human rotavirus strainHRV P43 (ECACC 99081301), prevention is conferred againstgastroenteritis or severe gastroenteritis caused by a rotavirus strainof a P[8] type and by a non- P[8] type, for example by a rotavirusstrain having a serotype selected from the list consisting of: P1, P2,P3, P4, P5, P6, P7, P9 and P11 types. In particular, the immunogeniccomposition described herein is capable of inducing an immune responseagainst, and/or provide protection against gastroenteritis or severegastroenteritis caused by, at least one, suitably all of the followingnon- P[8] type: P4, P6. In another embodiment, the composition iscapable of inducing an immune response to, and/or provide protectionagainst gastroenteritis or severe gastroenteritis caused by, a rotavirusstrain of a different G type and a different P type to that present inthe administered composition. Suitably the composition is also capableof inducing an immune response to, and/or provide protection againstgastroenteritis or severe gastroenteritis caused by, a G2P[4] rotavirusstrain.

The immunogenic composition of the invention is suitably provided in aform suitable for oral delivery.

In one embodiment, a vaccine according to the present invention isadministered as a liquid formulation. Suitably the liquid formulation isreconstituted prior to administration from at least the following twocomponents:

i) virus component;

ii) liquid diluent component.

In this embodiment, the virus component and the liquid diluent componentare normally present in separate containers, which may conveniently beseparate compartments of a single vessel, or separate vessels which canbe connected in such a way that the final vaccine composition isreconstituted without exposing it to the air.

Prior to reconstitution, the virus may be in a dry form or a liquidform. Suitably, in a rotavirus vaccine, the rotavirus is lyophilised.Lyophilised virus is more stable than virus in an aqueous solution. Thelyophilised virus may be suitably reconstituted using a liquid antacidcomposition to produce a liquid formulation. Alternatively thelyophilised virus may be reconstituted with water or aqueous solution,in which case the lyophilised virus composition suitably contains anantacid component.

In another embodiment, the immunogenic composition or vaccine of theinvention is a solid formulation, such as a lyophilised composition,suitably a lyophilised cake which is suitable for immediate dissolutionwhen placed in the mouth. Lyophilised formulations may conveniently beprovided in the form of tablets. In another aspect the inventionprovides a vaccine, such as a rotavirus vaccine, in the form of a quickdissolving tablet for oral administration.

The composition according to the invention may be provided in the formof an oral thin film. Accordingly, an oral thin film (“OTF”, also knownas a “dissolving film” or “oral drug strip”) may be used to administerthe immunogenic composition of the invention via absorption in the mouth(buccally or sublingually). Preferably, an OTF comprising an immunogeniccomposition of the invention is for buccal or sublingual administration.An OTF may be prepared, for example, by using hydrophilic polymers thatrapidly dissolve on the tongue or buccal cavity, delivering theimmunogenic composition to the systemic circulation via dissolution whencontact with liquid is made. According to one aspect of the inventionthere is provided an OTF comprising an immunogenic composition of theinvention.

A suitable amount of virus will normally be between 10⁴ and 10⁷ ffu pervaccine dose. A typical dose of vaccine may comprise 10⁵-10⁶ffu perdose, and multiple doses may be given over a period of time, for exampletwo doses given at a two-month interval. Rotavirus titer may also beexpressed in CCID₅₀ and it can be estimated in the context of thisinvention that a CCID₅₀ of 10^(6.0) is equivalent to a ffu of 10^(5.5)per dose. Benefits may however be obtained by having more than 2 doses,for example a 3 or 4 dose regimen, particularly in developing countries.The first dose can suitably be given to infants at from about 4 weeks toabout 14 or 15 weeks of age, suitably between 6 and 14 weeks of age. Theinterval between doses is at least 4 weeks but may be more or less thantwo months long, for example the second dose, and any subsequent dose ifappropriate, may be given one month or three months after the previousdose, depending on the local immunisation schedule. An optimal amount ofvirus for a single dose or for a multiple dose regimen, and optimaltiming for the doses, can be ascertained by standard studies involvingobservation of antibody titers and other responses in subjects.

Typically the volume of a dose of vaccine according to the inventionwill be 2.5 ml or lower, typically 0.2 ml to 2.0 ml. In a specificaspect of the invention, a suitable dose will normally be 1.5 ml orsuitably any volume smaller than 2.5 ml such as a volume of 2 ml orless, that is suitable for oral administration to babies or infants. Inparticular the dose volume will be such that the technical feasibilityof the formulation is possible and there is no detrimental effect on theimmunogenic potential of the formulation. Suitably the dose volume is0.5 ml to 1.5 ml, suitably approximately 1.0 ml to 1.5 ml, such as about1.3 ml or about 1.4 ml or about 1.5 ml.

In one aspect there is provided a kit comprising the immunogeniccomposition according to the invention and instructions for use of thekit.

The subject matter of and information disclosed within the publicationsand patents or patent applications mentioned in this specification areincorporated by reference in their entirety herein.

The present invention will now be further described by means of thefollowing non-limiting examples.

EXAMPLES

In all examples, the rotavirus concentration was 6.3 log₁₀ CCID/ml.

Example 1: Effect of Different Carboxylates, Different Divalent Cationsand Different Sugars/Polyols on the Thermostability of RotavirusFormulations

The effects of the following components on the thermostability of liquidrotavirus formulations were assessed:

Buffers: adipic acid, citric acid.

Divalent cation: no divalent cation, calcium chloride, magnesiumsulphate, zinc chloride, Sugar or Polyol: sucrose, glucose, maltose,trehalose dihydrate, fructose, α-cyclodextrin, sorbitol, dextransulfate.

Table 1 below provides details on each tested composition. Each testedcomposition had a volume of 1.4 ml and at a pH of 6.5. ‘Ch’ refers tochloride.

TABLE 1 Conc. Sugar/ Conc. Divalent Conc. Run Buffer (w/v) Polyol (w/v)cation (mM) 1 Citric acid 3.13% Sorbitol 30% Calcium Ch 15 mM 2 Citricacid 3.13% Sorbitol 30% Zinc Ch 15 mM 3 Citric acid 3.13% Maltose  9% Nodivalent 4 Citric acid 3.13% Glucose 30% Zinc Ch 15 mM 5 Citric acid3.13% Cyclodextrin  6% Zinc Ch 15 mM 6 Adipic 6.70% Glucose 30%Magnesium 15 mM acid 7 Citric acid 3.13% Sucrose 30% Magnesium 15 mM 8Citric acid 3.13% Dextran 4.80%   No divalent 9 Citric acid 3.13%Cyclodextrin  6% Magnesium 15 mM 10 Citric acid 3.13% Cyclodextrin  6%Calcium Ch 15 mM 11 Adipic 6.70% Sucrose 30% Zinc Ch 15 mM acid 12Citric acid 3.13% Maltose  9% Zinc Ch 15 mM 13 Citric acid 3.13% Glucose30% Calcium Ch 15 mM 14 Adipic 6.70% Trehalose 30% No divalent acid 15Adipic 6.70% Cyclodextrin  6% Calcium Ch 15 mM acid 16 Adipic 6.70%Dextran 4.80%   Magnesium 15 mM acid 17 Adipic 6.70% Maltose  9% Zinc Ch15 mM acid 18 Citric acid 3.13% Sorbitol 30% No divalent 19 Citric acid3.13% Glucose 30% No divalent 20 Citric acid 3.13% Fructose 30% Nodivalent 21 Citric acid 3.13% Fructose 30% Magnesium 15 mM 22 Adipic6.70% Maltose  9% Calcium Ch 15 mM acid 23 Citric acid 3.13% Maltose  9%Calcium Ch 15 mM 24 Adipic 6.70% Sorbitol 30% Calcium Ch 15 mM acid 25Adipic 6.70% Sucrose 30% Magnesium 15 mM acid 26 Adipic 6.70% Fructose30% No divalent acid 27 Adipic 6.70% Sucrose 30% No divalent acid 28Adipic 6.70% Cyclodextrin  6% Magnesium 15 mM acid 29 Citric acid 3.13%Glucose 30% Magnesium 15 mM 30 Citric acid 3.13% Sucrose 30% Zinc Ch 15mM 31 Citric acid 3.13% Cyclodextrin  6% No divalent 32 Adipic 6.70%Sorbitol 30% Magnesium 15 mM acid 33 Adipic 6.70% Sorbitol 30% Nodivalent acid 34 Adipic 6.70% Glucose 30% No divalent acid 35 Citricacid 3.13% Sorbitol 30% Magnesium 15 mM 36 Adipic 6.70% Dextran 4.80%  No divalent acid 37 Adipic 6.70% Cyclodextrin  6% No divalent acid 38Adipic 6.70% Fructose 30% Magnesium 15 mM acid 39 Citric acid 3.13%Maltose  9% Magnesium 15 mM 40 Adipic 6.70% Fructose 30% Calcium Ch 15mM acid 41 Adipic 6.70% Maltose  9% No divalent acid 42 Citric acid3.13% Trehalose 30% Zinc Ch 15 mM 43 Adipic 6.70% Dextran 4.80%  Calcium Ch 15 mM acid 44 Citric acid 3.13% Dextran 4.80%   Calcium Ch 15mM 45 Citric acid 3.13% Dextran 4.80%   Magnesium 15 mM 46 Citric acid3.13% Trehalose 30% No divalent 47 Adipic 6.70% Trehalose 30% Magnesium15 mM acid 48 Citric acid 3.13% Trehalose 30% Magnesium 15 mM 49 Adipic6.70% Trehalose 30% Calcium Ch 15 mM acid 50 Citric acid 3.13% Sucrose30% Calcium Ch 15 mM 51 Citric acid 3.13% Sucrose 30% No divalent 52Adipic 6.70% Cyclodextrin  6% Zinc Ch 15 mM acid 53 Citric acid 3.13%Dextran 4.80%   Zinc Ch 15 mM 54 Adipic 6.70% Glucose 30% Calcium Ch 15mM acid 55 Adipic 6.70% Maltose  9% Magnesium 15 mM acid 56 Control6.70% Sucrose 72% No divalent Rota 57 Citric acid 3.13% Fructose 30%Calcium Ch 15 mM 58 Citric acid 3.13% Fructose 30% Zinc Ch 15 mM 59Adipic 6.70% Sucrose 30% Calcium Ch 15 mM acid 60 Citric acid 3.13%Trehalose 30% Calcium Ch 15 mM

Six replicates of each formulation were tested. The thermostability ofeach formulation was assessed by measuring log FFU/ml of rotavirus afterstorage for 7 days at 45° C.

Out of the 59 non-control compositions, 14 compositions provided LOGffu/ml readings above limit of quantification (LOQ). The results ofthese tests are shown in FIG. 1. Each replicate of a formulation isshown with a different symbol (some data points are obscured by otherdata points). REF refers to a reference sample of bench-preparedROTARIX™ oral rotavirus vaccine (run 27 in Table 1 above). The REFcomposition differs from commercially-available ROTARIX™ in that the REFcomposition contains 30% w/v sucrose (as opposed to 72% sucrose) and hasa pH of 6.5 (as opposed to a pH of 7). ‘Control Rota’ and ‘Control Ro’refer to a control composition comprising 72% w/v sucrose, 6.7% w/vadipic acid and having a pH of 6.5.

Only the seven circled test formulations demonstrated a LOG ffu/ml aboveLOQ for all repeats. It can be seen from FIG. 1 that formulationsincorporating maltose, trehalose and α-cyclodextrin provided relativelypoor or inconsistent rotavirus thermostability.

These seven highlighted formulations are also shown for furthercomparison in FIG. 2. It can be seen from FIG. 2 that the formulationcomprising adipic acid, sucrose and calcium had the highest average LOGffu/ml (indicated by horizontal lines on FIG. 2) and the formulationcomprising adipic acid, sucrose and magnesium had the second highestaverage LOG ffu/ml. Both of these formulations had a higher average LOGffu/ml than the reference formulation (which comprised no divalentcation). The formulations comprising dextran or sorbitol, as opposed tosucrose, had average LOG ffu/ml readings which were lower than thereference. The formulations comprising citric acid, as opposed to adipicacid, had average LOG ffu/ml readings lower than the reference.

Example 2: Effect of Particular Amino Acids on the Thermostability ofRotavirus Formulations

The effect of adding particular amino acids (and calcium pantothenate)to rotavirus formulations comprising (a) sucrose, adipic acid andcalcium or (b) sucrose, adipic acid and magnesium was assessed.

Table 2 below provides details on each tested composition. Each testedcomposition had a volume of 1.4 ml and at a pH of 6.5. ‘Ch’ refers tochloride. ‘CTRL’ refers to no amino acid present.

TABLE 2 Buffer Conc. Conc. Divalent Conc. Run Type (w/v) Polyol (w/v)cation Conc. Amino acid (w/v) 1 Adipic 6.70% Sucrose 30% Magnesium 15CTRL — acid mM 2 Adipic 6.70% Sucrose 30% Magnesium 15 Aspartic acid0.028%  acid mM 3 Adipic 6.70% Sucrose 30% Magnesium 15 Proline 0.1%acid mM 4 Adipic 6.70% Sucrose 30% Magnesium 15 Threonine 0.1% acid mM 5Adipic 6.70% Sucrose 30% Magnesium 15 Valine 0.1% acid mM 6 Adipic 6.70%Sucrose 30% Magnesium 15 Histidine 0.1% acid mM 7 Adipic 6.70% Sucrose30% Magnesium 15 Methionine 0.1% acid mM 8 Adipic 6.70% Sucrose 30%Magnesium 15 Arginine 0.1% acid mM 9 Adipic 6.70% Sucrose 30% Magnesium15 Tyrosine 0.0014%   acid mM 10 Adipic 6.70% Sucrose 30% Magnesium 15Calcium 0.1% acid mM pantothénate 11 Adipic 6.70% Sucrose 30% Magnesium15 Cysteine 0.1% acid mM 12 Adipic 6.70% Sucrose 30% Magnesium 15 Sodium0.1% acid mM glutamate 13 Adipic 6.70% Sucrose 30% Magnesium 15 Glycine0.1% acid mM 14 Adipic 6.70% Sucrose 30% No divalent — CTRL — acid 15Adipic 6.70% Sucrose 30% No divalent — Aspartic acid 0.028%  acid 16Adipic 6.70% Sucrose 30% No divalent — Proline 0.1% acid 17 Adipic 6.70%Sucrose 30% No divalent — Threonine 0.1% acid 18 Adipic 6.70% Sucrose30% No divalent — Valine 0.1% acid 19 Adipic 6.70% Sucrose 30% Nodivalent — Histidine 0.1% acid 20 Adipic 6.70% Sucrose 30% No divalent —Methionine 0.1% acid 21 Adipic 6.70% Sucrose 30% No divalent — Arginine0.1% acid 22 Adipic 6.70% Sucrose 30% No divalent — Tyrosine 0.0014%  acid 23 Adipic 6.70% Sucrose 30% No divalent — Calcium 0.1% acidpantothénate 24 Adipic 6.70% Sucrose 30% No divalent — Cysteine 0.1%acid 25 Adipic 6.70% Sucrose 30% No divalent — Sodium 0.1% acidglutamate 26 Adipic 6.70% Sucrose 30% No divalent — Glycine 0.1% acid 27Citric 3.13% Sucrose 30% Calcium Ch 15 CTRL — acid mM 28 Citric 3.13%Sucrose 30% Calcium Ch 15 Aspartic acid 0.028%  acid mM 29 Citric 3.13%Sucrose 30% Calcium Ch 15 Proline 0.1% acid mM 30 Citric 3.13% Sucrose30% Calcium Ch 15 Threonine 0.1% acid mM 31 Citric 3.13% Sucrose 30%Calcium Ch 15 Valine 0.1% acid mM 32 Citric 3.13% Sucrose 30% Calcium Ch15 Histidine 0.1% acid mM 33 Citric 3.13% Sucrose 30% Calcium Ch 15Methionine 0.1% acid mM 34 Citric 3.13% Sucrose 30% Calcium Ch 15Arginine 0.1% acid mM 35 Citric 3.13% Sucrose 30% Calcium Ch 15 Tyrosine0.0014%   acid mM 36 Citric 3.13% Sucrose 30% Calcium Ch 15 Calcium 0.1%acid mM pantothénate 37 Citric 3.13% Sucrose 30% Calcium Ch 15 Cysteine0.1% acid mM 38 Citric 3.13% Sucrose 30% Calcium Ch 15 Sodium 0.1% acidmM glutamate 39 Citric 3.13% Sucrose 30% Calcium Ch 15 Glycine 0.1% acidmM 40 Adipic 6.70% Sucrose 30% Calcium Ch 15 CTRL — acid mM 41 Adipic6.70% Sucrose 30% Calcium Ch 15 Aspartic acid 0.028%  acid mM 42 Adipic6.70% Sucrose 30% Calcium Ch 15 Proline 0.1% acid mM 43 Adipic 6.70%Sucrose 30% Calcium Ch 15 Threonine 0.1% acid mM 44 Adipic 6.70% Sucrose30% Calcium Ch 15 Valine 0.1% acid mM 45 Adipic 6.70% Sucrose 30%Calcium Ch 15 Histidine 0.1% acid mM 46 Adipic 6.70% Sucrose 30% CalciumCh 15 Methionine 0.1% acid mM 47 Adipic 6.70% Sucrose 30% Calcium Ch 15Arginine 0.1% acid mM 48 Adipic 6.70% Sucrose 30% Calcium Ch 15 Tyrosine0.0014%   acid mM 49 Adipic 6.70% Sucrose 30% Calcium Ch 15 Calcium 0.1%acid mM pantothénate 50 Adipic 6.70% Sucrose 30% Calcium Ch 15 Cysteine0.1% acid mM 51 Adipic 6.70% Sucrose 30% Calcium Ch 15 Sodium 0.1% acidmM glutamate 52 Adipic 6.70% Sucrose 30% Calcium Ch 15 Glycine 0.1% acidmM

Six replicates of each formulation were tested: three replicates in afirst analytical run and three replicates in a second analytical run.The thermostability of each formulation was assessed by measuring LOGffu/ml of rotavirus after storage for 10 days at 45° C.

The average LOG ffu/ml for all formulations containing adipic acid andno divalent cation was lower than the average LOG ffu/ml for allformulations containing adipic acid and magnesium or calcium. Theaverage LOG ffu/ml for all formulations containing citric acid andcalcium was lower still (data not shown).

Calcium Ion- and Adipic Acid-Containing Formulations

The results of the assessment in respect of adipic acid and calciumion-containing formulations are shown in FIG. 3. Data from the firstanalytical run is denoted by the + symbol and data from the secondanalytical run is denoted by the * symbol. It can be seen from thisfigure that the tested amino acids provide varying levels of rotavirusthermostability enhancement. In the context of these formulationscomprising calcium ions, histidine provided a particularly high averagelevel of thermostability enhancement, followed by arginine.

Magnesium Ion- and Adipic Acid-Containing Formulations

The results of the assessment in respect of adipic acid and magnesiumion-containing formulations are shown in FIG. 4. Data from the firstanalytical run is denoted by the + symbol and data from the secondanalytical run is denoted by the * symbol. It can be seen from thisfigure that the tested amino acids provide varying levels of rotavirusthermostability enhancement. In the context of these formulationscomprising magnesium ions, arginine provided the highest average levelof thermostability enhancement.

Example 3: Stability of Rotavirus Formulations Comprising Arginineand/or Histidine After Storage for 4, 8 and 10 Weeks at 37° C.

New batches of the formulations from Example 2 comprising histidineand/or arginine were prepared and stored at 37° C. for 4, 8 and 10 weeks(indicated as 1 month, 2 months, and 2.5 months, respectively, in FIG.5), alongside reference formulations comprising (a) calcium chloride butno amino acids and (b) bench-prepared Rotarix vaccine (which comprisesno calcium ions and no added amino acids). The LOG ffu/ml of each ofthese formulations at each of these timepoints is shown in FIG. 5. Inparallel, a control formulation corresponding to each formulation wasalso assayed at 4° C. The difference in LOG ffu/ml between each controlreading and the 10 week time point for each formulation is shown in abox below each data set. The viral titer of the reference formulationsafter 10 weeks is highlighted by the horizontal line. It can be seenfrom FIG. 5 that the addition of histidine and/or arginine to theseformulations resulted in a surprising improvement in rotavirus stabilitycompared to the reference formulations.

Example 4: Stability of Rotavirus Formulations Further ComprisingVarious Antioxidants and Proteins

Formulations according to Examples 1 to 3 above were modified byaddition of one of the following further components each: vitamin Esuccinate (VES), heparin, monothioglycerol (MTG), lactalbuminhydrolysate, albumin, or β casein.

Each formulation contained quantities of each component as set out inTable 3 in a volume of 1.4 ml and at a pH of 6.5. Six replicates of eachformulation were tested. The thermostability of each formulation wasassessed by measuring LOG ffu/ml of rotavirus after storage for 10 daysat 45° C.

TABLE 3 Protein or Buffer Conc. Conc. Divalent Amino anti- No. Type(w/v) Polyol (w/v) cation Conc. Acid Conc. oxidant Conc. 1 Adipic 6.70%Sucrose 30% Calcium 15 Arginine 0.10% VES 0.9 mM acid mM 2 Adipic 6.70%Sucrose 30% Calcium 15 Histidine 0.10% VES 0.9 mM acid mM 3 Adipic 6.70%Sucrose 30% CTRL — CTRL — VES 0.9 mM acid 4 Adipic 6.70% Sucrose 30%Calcium 15 Arginine 0.10% Heparin  0.10% acid mM 5 Adipic 6.70% Sucrose30% Calcium 15 Histidine 0.10% Heparin  0.10% acid mM 6 Adipic 6.70%Sucrose 30% CTRL — CTRL — Heparin  0.10% acid 7 Adipic 6.70% Sucrose 30%Calcium 15 Arginine 0.10% MTG  0.25% acid mM 8 Adipic 6.70% Sucrose 30%Calcium 15 Histidine 0.10% MTG  0.25% acid mM 9 Adipic 6.70% Sucrose 30%CTRL — CTRL — MTG  0.25% acid 10 Adipic 6.70% Sucrose 30% Calcium 15Arginine 0.10% Lactal-  0.10% acid mM bumin hydrolysate 11 Adipic 6.70%Sucrose 30% Calcium 15 Histidine 0.10% Lactal-  0.10% acid mM buminhydrolysate 12 Adipic 6.70% Sucrose 30% CTRL — CTRL — Lactal-  0.10%acid bumin hydrolysate 13 Adipic 6.70% Sucrose 30% Calcium 15 Arginine0.10% Albumin  0.10% acid mM 14 Adipic 6.70% Sucrose 30% Calcium 15Histidine 0.10% Albumin  0.10% acid mM 15 Adipic 6.70% Sucrose 30% CTRL— CTRL — Albumin  0.10% acid 16 Adipic 6.70% Sucrose 30% Calcium 15Arginine 0.10% B casein 0.036% acid mM 17 Adipic 6.70% Sucrose 30%Calcium 15 Histidine 0.10% B casein 0.036% acid mM 18 Adipic 6.70%Sucrose 30% CTRL — CTRL — B casein 0.036% acid 19 Adipic 6.70% Sucrose30% Calcium 15 Arginine 0.10% CTRL — acid mM 20 Adipic 6.70% Sucrose 30%Calcium 15 Histidine 0.10% CTRL — acid mM 21 Adipic 6.70% Sucrose 30%CTRL — CTRL — CTRL — acid 22 CTRL 6.70% Sucrose 72% — — — — — — 72%stressed

The results are shown in FIG. 6. Boxes indicate different groupings offormulations. It can be seen from FIG. 6 that, in particular, theaddition of albumin generally enhanced the thermostability of theseformulations on average. The enhancing effect was visible both in thepresence and in the absence of arginine, histidine and/or calcium.

Example 5: Further Enhancement of Rotavirus Formulation Thermostability

A suite of tests were performed in an attempt to identify a quadraticmodel of the impact of seven parameters (presence and concentration ofsucrose, calcium, histidine, arginine, TPGS and albumin, and pH level)on thermostability of rotavirus formulations.

46 test formulations were prepared alongside 7 control formulations and3 placebo formulations. Each formulation is labelled with a number (#).Control formulations (#1, 2, 32, 33, 34, 35 and 37) were assayed afterstorage for 14 days at 4° C. Placebo formulations contained norotavirus. 50 mL of each formulation was prepared and three replicatesof 1.4 ml of each formulation were tested for stability. In eachformulation, the concentration of adipic acid was 6.7% w/v. Table 4below provides details on every formulation. Percentages areweight/volume.

TABLE 4 Albumin Active/ Sucrose Calcium Magnesium His Arg TPGS (r-HSA) #Placebo (%) (mM) (mM) (%) (%) (mM) (%) pH 1 Active 72 0 0 0 0 0 0 6.5 2Active 72 15 0 0 0.1 0 0 6.5 3 Placebo 72 15 0 0 0.1 0 0 6.5 4 Active 725 0 0 0 0 0 7 5 Active 72 5 0 0 0 2 0.2 7 6 Active 72 5 0 0 0.1 0 0 6 7Active 72 5 0 0 0.2 2 0 7 8 Active 72 5 0 0.1 0 0 0.2 6 9 Active 72 5 00.2 0 1 0.1 7 10 Active 72 5 0 0.2 0 2 0 6 11 Active 72 5 0 0.2 0.1 00.2 7 12 Active 72 5 0 0.2 0.2 1 0 6.5 13 Active 72 5 0 0.2 0.2 2 0.2 614 Active 72 10 0 0 0.2 1 0.2 7 15 Active 72 10 0 0.1 0.1 2 0 7 16Active 72 10 0 0.2 0.2 0 0.1 6 17 Active 72 15 0 0 0 0 0.2 7 18 Active72 15 0 0 0 2 0.2 6 19 Active 72 15 0 0 0.2 0 0 7 20 Active 72 15 0 00.2 2 0 6 21 Active 72 15 0 0.1 0.2 0 0.2 6.5 22 Active 72 15 0 0.2 0 00 6 23 Active 72 15 0 0.2 0 2 0 7 24 Active 72 15 0 0.2 0.1 1 0.2 6 25Active 72 15 0 0.2 0.2 2 0.2 7 26 Active 51 5 0 0 0.2 0 0.2 6 27 Active51 5 0 0.2 0.2 0 0 7 28 Active 51 10 0 0.1 0.1 1 0.1 6.5 29 Active 51 100 0.2 0 2 0.2 6.5 30 Active 51 15 0 0 0 1 0 6 31 Active 51 15 0 0 0.1 20.1 7 32 Active 30 15 0 0.1 0 0 0 6.5 33 Active 30 15 0 0 0.1 0 0 6.5 34Active 30 15 0 0 0.1 1 0 6.5 35 Active 30 5 0 0 0 0 0 6 36 Placebo 30 50 0 0 0 0 6 37 Active 30 5 0 0 0 0 0 7 38 Placebo 30 5 0 0 0 0 0 7 39Active 30 5 0 0 0 0 0.2 7 40 Active 30 5 0 0 0 2 0.2 6 41 Active 30 5 00 0.2 0 0.1 6.5 42 Active 30 5 0 0 0.2 2 0 6 43 Active 30 5 0 0.2 0 0 06 44 Active 30 5 0 0.2 0 2 0 7 45 Active 30 5 0 0.2 0.2 0 0.2 6 46Active 30 5 0 0.2 0.2 2 0.2 7 47 Active 30 10 0 0 0.2 1 0 7 48 Active 3015 0 0 0 0 0.2 6 49 Active 30 15 0 0 0 2 0 6.5 50 Active 30 15 0 0 0.2 20.2 6 51 Active 30 15 0 0.1 0 2 0.2 7 52 Active 30 15 0 0.1 0.2 0 0 6 53Active 30 15 0 0.2 0 0 0 7 54 Active 30 15 0 0.2 0 2 0.1 6 55 Active 3015 0 0.2 0.2 0 0.2 7 56 Active 30 15 0 0.2 0.2 2 0 6.5

The mean thermostability of each test formulation was assessed bymeasuring LOG ffu/ml of rotavirus after storage for 14 days at 45° C.

The results of the 14 day storage tests are shown in FIGS. 7-12. FIG. 7provides thermostability results in respect of all tested parametersafter 14 days storage at 45° C. and FIGS. 8-12 each more closelyillustrate particular aspects of these same data. Control formulationsare denoted by plus sign (+). Tables 5, 6 and 7 below repeat theformulation details in Table 4 but set out in line with the arrangementof FIG. 7.

TABLE 5 SUCROSE 72% w/v Sucrose Albumin Histidine Arginine TPGS Calcium(% w/v) (% w/v) (% w/v) (% w/v) (mM) (mM) pH 72 0.2 0.2 0.2 2 15 7 5 60.1 1 15 6 0 5 7 0.1 0.2 0 15 6.5 0 5 6 0 0.2 1 10 7 0 2 15 6 5 7 0 15 70.1 0.2 0.2 0 10 6 0 1 5 7 0 0.2 0.2 1 5 6.5 0 2 15 7 5 6 0 15 6 0.1 0.12 10 7 0 0.2 2 15 6 0.2 2 5 7 0 15 7 0.1 0 15 6.5 5 6 0 0 5 7 0 6.5

TABLE 6 SUCROSE 51% w/v Sucrose Albumin Histidine Arginine TPGS CalciumpH 51 0.2 0.2 0 2 10 6.5 0 0.2 0 5 6 0.1 0.1 0.1 1 10 6.5 0 0.1 2 15 7 00.2 0.2 0 5 7 0 0 1 15 6

TABLE 7 SUCROSE 30% w/v Sucrose Albumin Histidine Arginine TPGS Calcium% w/v % w/v % w/v % w/v mM mM pH 30 0.2 0.2 0.2 2 5 7 0 15 7 5 6 0.1 0 215 7 0 0.2 2 15 6 0 2 5 6 0 15 6 5 7 0.1 0.2 0 2 15 6 0 0.2 0 5 6.5 00.2 0.2 2 15 6.5 0 2 5 7 0 15 7 5 6 0.1 0.2 0 15 6 0 0 15 6.5 0 0.2 2 56 1 10 7 0.1 1 15 6.5 0 15 6.5 0 2 15 6.5 0 5 7 6

It can be seen from FIG. 7 that, in particular, the formulationscomprising low (30% w/v) sucrose, calcium ions, plus histidine and/orarginine were especially effective, maintaining high titres after 14days storage. The two most effective formulations (circled) providingthe highest viral titres contained (a) 30% w/v sucrose, 15 mM calciumions, 0.1% w/v histidine, 0% w/v arginine, 2 mM TPGS and 0.2% w/vrecombinant human serum albumin; and (b) 30% w/v sucrose, 5 mM calciumions, 0.2% w/v histidine, 0.2% w/v arginine, 2 mM TPGS and 0.2% w/vrecombinant human serum albumin; both formulations having a pH of 7.

Interactions Between Components

FIG. 8 illustrates the interaction between albumin and sucrosespecifically. It can be seen from FIG. 8 that at both 72% and 51% w/vsucrose, the addition of albumin to these formulations made no impact ora small detrimental impact to mean viral titre. In contrast, at 30% w/vsucrose, the presence of albumin resulted in higher mean viral tires. Itappears that the thermostability improvements to these formulationsprovided by the addition of albumin are negated by the addition of highlevels of sucrose.

FIG. 9 illustrates the interaction between histidine and sucrosespecifically. It can be seen from FIG. 9 that higher quantities ofhistidine generally provide improved average viral titres, regardless ofsucrose concentration at 30, 51 or 72% w/v. However, the highest viraltitre readings were achieved in formulations with histidine present at30% w/v sucrose concentration.

FIG. 10 illustrates the interaction between TPGS and albuminspecifically. It can be seen from FIG. 10 that higher levels of bothTPGS and albumin both generally improved thermostability.

FIG. 11 illustrates the impact of increasing levels of arginine on thetested formulations. It can be seen from FIG. 11 that higher levels ofarginine generally improved mean viral titre.

FIG. 12 illustrates the impact of pH variation on the testedformulations. It can be seen from FIG. 12 that from amongst pH 6, 6.5and 7, the highest viral titres are generally achieved at pH 6.5.

Example 6: A Highly Thermostable Formulation

A composition was formulated comprising 6.7% adipate buffer, 10% w/vsucrose, 5 mM calcium ions, 0.2% w/v histidine, 0.2% w/v arginine, 0.2%w/v recombinant human serum albumin (rHSA), 1 mM TPGS and having a pH of6.5. The composition was produced in two separate lots. Thethermostability of this formulation was assessed alongside commerciallyavailable Rotarix over a period of 8 weeks storage at 40° C.Commercially available Rotarix differs from the bench-prepared Rotarixused in Examples 1-5 in the manner detailed in Example 1.

Titre readings were taken at the start of the assay (TO) and every weekthereafter. The results are shown in FIG. 13. In FIG. 13, for each oflot 1, lot 2 and the commercial control, the columns from left to rightare TO, T1w 40° C., T2w 40° C., T3w 40° C., T4w 40° C., T5w 40° C., T6w40° C., T7w 40° C. and T8w 40° C. It can be seen from FIG. 13 that after7 weeks, this exemplary formulation lost approximately 1 log of viraltitre, while the viral titre dropped below the limit of quantificationin the Rotarix control after the same time period.

1.-24. canceled
 25. An immunogenic composition comprising: a viralantigen, a sugar and/or polyol, an adipate buffer, calcium ions and/ormagnesium ions, and one or more positively charged amino acids.
 26. Theimmunogenic composition according to claim 25, wherein the viral antigenis a rotavirus antigen.
 27. The immunogenic composition of claim 26,wherein the rotavirus antigen is a live attenuated rotavirus.
 28. Theimmunogenic composition of claim 25, wherein the one or more positivelycharged amino acids are histidine and/or arginine.
 29. The immunogeniccomposition according to claim 25, wherein the pH of the immunogeniccomposition is between about pH 6.0 and about pH 7.0.
 30. Theimmunogenic composition according to claim 25, wherein said compositionhas a level of thermostability such that, after storage of thecomposition for 2.5 months at 37° C., the composition has a maximumvirus titer loss of 1.5, as expressed in log₁₀ ffu/mL as compared to thecomposition's initial virus titer before storage.
 31. The immunogeniccomposition according to claim 25 wherein the rotavirus is present at atitre ranging from about 1×10⁵ to about 1×10⁸pfu/mL; the concentrationof the sugar and/or polyol is about 1% w/v to about 70% w/v, preferablyno more than 30% w/v; the adipate buffer is present at a concentrationof about 1% to about 20% w/v: the calcium ions and/or magnesium ions arepresent at a concentration from about 1 mM to about 15 mM; and the oneor more amino acid is present at a concentration of at least 0.01% w/v,preferably of at least 0.15% w/v.
 32. The immunogenic compositionaccording to claim 25 wherein the sugar and/or polyol is sucrose orsorbitol, preferably sucrose.
 33. The immunogenic composition accordingto claim 25 further comprising a protein, preferably albumin at aconcentration of at least 0.05% w/v, more preferably human serumalbumin, for example recombinant human serum albumin.
 34. Theimmunogenic composition according to claim 25 comprising vitamin Esuccinate or TPGS, preferably at a concentration of at least 0.5 mM. 35.The immunogenic composition according to claim 25 wherein theimmunogenic composition is provided in a dose volume of 0.2 ml to 2.0ml.
 36. The immunogenic composition according to claim 25 wherein theimmunogenic composition is suitable for oral administration.
 37. Theimmunogenic composition according to claim 25 wherein the immunogeniccomposition is in the form of a liquid.
 38. The immunogenic compositionaccording to claim 25 wherein the immunogenic composition is in the formof a tablet.
 39. The immunogenic composition according to claim 25wherein the composition is in the form of an oral thin film.
 40. Theimmunogenic composition according to claim 25, which is a vaccine.
 41. Apharmaceutical composition comprising the immunogenic compositionaccording to claim 25 and a pharmaceutically acceptable excipient. 42.An immunogenic composition or pharmaceutical composition according toclaim 25 for use as a medicament.
 43. The immunogenic composition orpharmaceutical composition according to claim 42 for use in theprevention of rotavirus infection.
 44. Use of the immunogeniccomposition or pharmaceutical composition according to claim 25 in themanufacture of a medicament for use in the prevention of rotavirusinfection.
 45. A method for the prevention of rotavirus infectioncomprising administering the immunogenic composition or pharmaceuticalcomposition of claim 25 to a subject having or at risk of rotavirusinfection.
 46. The immunogenic composition or pharmaceutical compositionfor use, the use or the method according to claim 25, wherein therotavirus infection is rotavirus infection in a human, preferably in ahuman infant.
 47. A kit comprising the immunogenic composition orpharmaceutical composition according to claim 25 and instructions foruse of the kit.
 48. A method for the preparation of an immunogeniccomposition according to claim 25, comprising admixing a rotavirus, asugar and/or polyol, an adipate buffer, calcium ions and/or magnesiumions, and histidine and/or arginine.